Materials for enhancing staining of biopolymers in matrices

ABSTRACT

Disclosed is a method for detecting biopolymers in a matrix. More specifically, the invention involves  
     (a) contacting the matrix with a sensitizing reagent comprising one or more optionally substituted heteroaromatic compounds;  
     (b) contacting the matrix with one or more reduceable metal salts to stain said biopolymer; and  
     (c) detecting the stained biopolymer.  
     The invention also relates to compositions for carrying out the invention and compositions made according to the invention. The invention also relates to kits for carrying out the methods of the invention.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention is in the field of metallic stains for biopolymerssuch as proteins, polypeptides and nucleic acid molecules when fixed insynthetic matrixes.

[0003] 2. Related Art

[0004] Gel electrophoresis is a commonly used analytical technique inbiochemistry and related fields of study for the separation of nucleicacids, polypeptides, proteins and oligosaccharides. A sample of interestis placed in a matrix and exposed to an electric field which causesvarious components to migrate and separate into distinct bands dependentupon the molecular weight, charge, and other physical properties of themolecules. After electrophoresis has ended, the pattern of migration isnot typically decipherable because the majority of molecules have nointegral choromophores or fluorophores by which they may be visualized.Numerous methods have been developed to visualize the exact locations ofthe molecules of interest within a matrix while leaving the blank areasof the matrix virtually unstained. These include the Coomassie BrillantBlue dyes, ethidium bromide and Ponceau S stains. Silver staining wasdeveloped to increase the sensitivity over that achieved with thesedyes. One of the earliest and widely used silver staining technique wasreported by Merril et al., Meth. Enzymol. 96:230 (1983). In this report,an electrophoretic matrix, specifically polyacrylamide, is immersed ineither an acid or an acid/alcohol solution for about one hour to fix theproteins within the matrix. The matrix is then washed typically forabout thirty minutes. The matrix is then soaked for about five minutesin a dichromic acid solution to oxidize the protein. Next, the gels aresoaked in a silver nitrate solution for twenty minutes and then rinsedwith a sodium carbonate/formaldehyde buffer to reduce silver ions boundto proteins and nucleic acids. A silver pattern is allowed to developand then stopped by the addition of acetic acid. The pattern is thenanalyzed either by direct visualization or by instrumental techniques.

[0005] The method of Merril et al. was simplified by Oakley et al.,Anal. Biochem.105: 361 (1980). Electrophoresed gels were treated withunbuffered glutaraldehyde to cross-link proteins. Following rinsing, thegels were treated with a silver nitrate/ammonium hydroxide/sodiumhydroxide solution. Finally, a solution of formaldehyde in citric acidis used to reduce silver ion to silver and visualize the bands withinthe gel matrix.

[0006] Glutaraldehyde has been employed by many laboratories to lowerthe limit of detection of proteins by silver staining (Rabilloud, T.,Electrophoresis 11:785-794 (1990); Rabilloud, T., Cell. Mol. Biol.40:57-75(1994)). The exact mechanism by which this compound enhancessensitivity has not been evaluated extensively, but it is thought tofunction by first binding to free amino groups in the protein to form aSchiff base leaving a free aldehyde function. Free amino groups arefound at the N-terminus of the protein and also on the side chain oflysines and arginines. This aldehyde may reduce silver in the proteinzone leading to an initiation of silver metal deposition. Silver metalis thought to catalyze further reduction of silver ion to silver so thenet result is to increase the rate of silver deposition adjacent to theprotein. An example of such a product employing glutaraldehydesensitization is the SILVERXPRESS silver stain sold by InvitrogenCorporation, Carlsbad, Calif.

[0007] Use of glutaraldehyde, though it improves sensitivity of thestain to low levels of proteins, has an undesirable effect when furtheranalysis of the gel by mass spectrometry is desired. Scheler et al.,Electrophoresis 19:918-927 (1998). A widely used technique for theanalysis of proteins by mass spectrometry is to cleave the protein intofragments with the enzyme trypsin. The fragments of a protein created bytryptic cleavage are readily predicted because the enzyme preferentiallycleaves the amide bond immediately after a basic amino acid such aslysine or arginine. If the amino group of lysine or arginine is bound ina Schiff base by glutaraldehyde, it is no longer a cleavage site fortrypsin. In addition, when an initial complex is formed between theglutaraldehyde molecule and the protein, the remaining free aldehydegroup is available to condense with other amino groups thereby producingcrosslinked proteins. This property is what makes glutaraldehyde such aneffective fixative for histological applications. However bycrosslinking peptides and proteins containing amino groups in a randomfashion, not only are the potential trypsin cleavage sites blocked,crosslinked peptides and peptides are randomly created whose molecularweight may not be predicted from an analysis of the protein primarystructure. As a result, there is a reduced abundance of many fragmentswhose molecular weight would be diagnostic for the identity of theprotein. Although other enzymes can be used to fragment the protein, thespecificity of trypsin makes it a favored choice.

[0008] U.S. Pat. No. 4,405,720 discloses a silver staining method forpolypeptides in gels comprising photo-reversing the polypeptide-gel bytreatment with an oxidizing reagent, forming a latent stain image bytreating the polypeptide-gel with a reduceable metal salt termed aphotosensitive salt, and developing the stain image by treating thepolypeptide gel with a reducing agent. Examples of such photosensitivesalts include salts of silver, gold, platinum, palladium and/or iridium.

[0009] U.S. Pat. No. 4,468,466 discloses a silver staining methodcomprising treatment with the reducing agent dithiothreitol followed bytreatment with a silver salt and actuating radiation. According to thispatent, dithiothreitol acts as a reducing agent to effect photoreversaland avoid silver staining of non-proteins.

[0010] U.S. Pat. No. 4,703,016 discloses a silver staining procedure forproteins and DNA. The process comprises fixing a protein on a membranein cupric acetate solution; contacting the membrane with a solutioncomprising acetic acid, sodium chloride, and citric acid; contacting themembrane with a solution comprising acetic acid and silver nitrate andirradiating with a light source; contacting the membrane with a solutioncomprising acetic acid, sodium chloride and citric acid; transferringthe membrane back to the silver nitrate solution and irradiating themembrane; developing the image by transferring to a solution comprisinghydroquinone and formaldehyde; washing with water, contacting withsodium thiosulfate; and then washing with water.

[0011] U.S. Pat. No. 4,575,452 discloses methods of detection ofproteins and nucleic acids in a matrix comprising fixing the proteinsand nucleic acids in the matrix with aromatic sulfonic acid compoundshaving tertiary amines and N,N′-di-(9-acridyl)-diaminoalkylenecompounds. Particular examples of such compounds include4,4′-[1,4-phenylenebis(2,5-oxazolediyl)]-bisbenzene-sulfonic acid,4,4′-[1,4-phenylenebis(4-methyl-2,5-oxazolediyl)]bisbenzene-sulfonicacid,2,2′-(2,5-thiophenediyl)bis[5-(1,1-dimethylethyl)-7-benzoxazole-sulfonicacid, N,N,N-trimethyl-2-phenyl-5-(4-sulfophenyl)-4-oxazolemethanamoniumhydroxide,2,2′-(1,4-phenylene)bis[N,N,N-trimethyl-5-(4-sulfophenyl)]-4-oxazolemethanamoniumhydroxide, and N,N′-di-(9-acridyl)-1,6-diaminohexane.

SUMMARY OF THE INVENTION

[0012] The invention relates in part to the discovery that it ispossible to provide a sensitivity of detection comparable or better thanprovided by glutaraldehyde fixation prior to staining without causingcrosslinking of peptides or blockage of trypsin cleavage sites.

[0013] It has also been discovered that compounds possessing an aromaticstructure containing heteroatoms capable of binding to silver ion can beused to improve the sensitivity of silver staining without impeding thecleavage of the protein by trypsin.

[0014] The present invention relates to a method for detecting abiopolymer in a matrix, comprising:

[0015] (a) contacting the matrix with a sensitizing reagent comprisingone or more optionally substituted heteroaromatic compounds;

[0016] (b) contacting the matrix with one or more reduceable metal saltsto stain said biopolymer; and

[0017] (c) detecting the stained biopolymer.

[0018] The present invention further relates to the analysis by massspectrometry of the biopolymer detected according to the presentinvention. In this embodiment, the biopolymer is recovered from thematrix, cleaved with a cleaving reagent, and subjected to massspectrometric analysis. In a preferred embodiment, the biopolymer is aprotein or peptide and the cleaving reagent comprises trypsin. Thus, ina further preferred embodiment, the invention relates to a method foridentifying a protein or peptide in a matrix, comprising:

[0019] (a) contacting the matrix with a sensitizing reagent comprisingone or more optionally substituted heteroaromatic compounds;

[0020] (b) contacting the matrix with one or more reduceable metal saltsto stain said protein or peptide;

[0021] (c) detecting the stained protein or peptide;

[0022] (d) carrying out a cleavage reaction on the protein or peptide togive fragments; and

[0023] (e) carrying out a mass spectrometric analysis on said fragmentsthereby identifying the protein or peptide.

[0024] The present invention also includes a kit for the detection ofbiopolymers comprising one or more components selected from the groupconsisting of

[0025] (a) a sensitizing reagent comprising one or more optionallysubstituted heteroaromatic compounds;

[0026] (b) one or more reduceable metal salts;

[0027] (c) one or more developer solutions comprising a reducing agent;

[0028] (d) one or more stopper solutions which prevent further reduction

[0029] (e) of the reduceable metal salts;

[0030] (f) one or more contrast enhancing agents;

[0031] (g) one or more buffers;

[0032] (h) one or more fixing reagents;

[0033] (i) one or more cleaving reagents;

[0034] (j) one or more biopolymers;

[0035] (k) one or more matrixes; and

[0036] (l) one or more indicators which are sensitive to pH changes.

[0037] The invention also relates to compositions for carrying out themethods of the invention as well as the compositions made according tothe invention. Such compositions of the invention may comprise one ormore of the components of the kit listed above.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Techniques for electrophoretically separating proteins,polypeptides and nucleic acids in a matrix are well known. In gelelectrophoresis, the molecules are separated into bands according to therate at which an imposed electric field causes them to migrate through amatrix. A particularly preferred matrix useful in the present inventionis polyacrylamide gel. Other useful matrices include agarose, paper,cellulose acetate, nitrocellulose, etc.

[0039] Examples of biopolymers that may be detected according to thepresent invention include nucleic acid molecules (e.g. DNA, RNA, hybridDNA-RNA, DNA and RNA derivatives such as phosphothioates and mixedbackbone derivatives), proteins, peptides and the like.

[0040] To fix biopolymers to a matrix, the matrix may be contacted witha fixing reagent or treated with microwave radiation. Such fixingreagents function to immobilize the biopolymer to the matrix and alsoremove substances which may interfere with the staining procedure. Suchfixing reagents include any conventional fixing reagents such as, forexample, glutaraldehyde, oxides of heavy metals such as mercury, leadand osmium, formaldehyde, paraformaldehyde, trichloroacetic acid andacetic acid. In another embodiment, the fixing reagent may be anaromatic sulfonic acid compound or a N,N′-di-(9-acridyl)-diaminoalkylenecompound disclosed in U.S. Pat. No. 4,575,452. In a preferredembodiment, the fixing reagent comprises an aqueous solution of anorganic acid and a lower alcohol containing 1-4 carbon atoms. A morepreferred fixing reagent comprises a lower alcohol such as methanol,ethanol, propanol, or isopropanol optionally with a commonly employedorganic acid. Such organic acids include without limitation acetic acid,citric acid, sulfosalicylic acid and trichloroacetic acid. In a mostpreferred embodiment, the fixing reagent consists of about 40% ethanol,about 10% acetic acid and about 50% distilled water by volume.Typically, a matrix may be fixed by immersion into a fixing reagent, andthe immersion in fresh fixing reagent may be repeated up to 3 times.

[0041] Incubation time with the fixing reagent is determined empiricallyand depends on the thickness of the matrix and the temperature at whichthe incubation is conducted. Enhancement in the rate of fixing may beachieved by heating the matrix submerged in the fixing solution, e.g.using a microwave generator. For room temperature fixations using a 8cm×8 cm×0.1 cm gel, the optimum fixing time is about 60 minutes on aplatform rotator at 60 revolutions/min. One of ordinary skill in the artcan determine the optimum time and temperature for fixing a given matrixwith no more than routine experimentation.

[0042] The matrix may then be treated with a washing solution to removeas much of the free acid as possible. The washing solution may comprisewater, about 10%-30% aqueous ethanol/methanol or about 10-30% aqueousethanol/methanol, further comprising a buffer to neutralize residualacid in the matrix. A preferred wash is 30% ethanol in water. Typicalincubation time for the matrix in the wash solution is about 10 min. Ifmicrowave heating is employed a shorter time of 2-5 min is preferred. Ina preferred embodiment, the matrix is immersed in the washing solutionfor a time sufficient to remove agents that adversely affect staining.

[0043] The matrix is then contacted with a sensitizing reagent. Thesensitizing reagent comprises at least one optionally substitutedheteroaromatic compound, preferably having at least two heteroatoms andbeing water soluble and, optionally, one or more contrast enhancingagents and buffers.

[0044] Optionally substituted heteroaromatic compounds having at leasttwo heteroatoms include those groups having 5 to 24 ring atoms; 6, 10 or14 π electrons shared in a cyclic array; and containing carbon atoms and2, 3 or 4 oxygen, nitrogen and/or sulfur atoms. Examples of suchheteraromatic compounds include without limitation thianthrene,phenoxathiin, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine,indazole, purine, phthalzine, naphthyridine, quinoxaline,1,4-dihydroquinoxaline-2,3-dione, quinozaline, cinnoline, pteridine,β-carboline, perimidine, phenanthroline, phenazine, isothiazole,phenothiazine, isoxazole, furazan, phenoxazine,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazole, benzimidazole, andbenzothiazole. Optional substitutents include 1, 2, 3, or 4 alkyl, halo,haloalkyl, nitro, amino, cyano, isocyano, hydroxy, thiol, alkoxy,sulfonyl, carboxy, optionally substituted aryl and optionallysubstituted heteroaromatic groups. Preferred substituents include thosewhich impart water solubility to the compound, e.g. amino, hydroxy,sulfonyl and carboxy groups. Heteroaromatic groups include thoseheteroaromatic groups listed above as well as thienyl, benzothienyl,naphthothienyl, furyl, pyranyl, chromanyl, isochromanyl, chromenyl,pyrrolyl, pyridyl, indolyl, quinolinyl, isoquinolinyl, coumarinyl, andcarbazolyl groups and the like.

[0045] Useful alkyl groups are C₁₋₂₀ alkyl groups including methyl,ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, hexyl,octyl, decyl, dodecyl, octadecyl groups and the like. Lower alkyl groupsare C₁₋₄ alkyl groups.

[0046] Useful halo groups include fluoro, chloro, bromo and iodo groups.

[0047] Useful haloalkyl groups include chloromethyl, bromomethyl,trifluoromethyl, trichloromethyl, pentachloromethyl, andpentafluoromethyl.

[0048] Useful amino groups include —NH₂ as well as mono- anddialkylamino groups such as methylamino, dimethylamino, ethylamino,propylamino, isopropylamino, butylamino and the like.

[0049] Useful aryl groups include are C₆₋₁₄ aryl, especially C₆₋₁₀ aryl.Typical C₆₋₁₄ aryl groups include phenyl, naththyl, phenanthryl,anthracyl, indenyl, biphenyl, biphenylenyl and fluorenyl groups.

[0050] Preferred optionally substituted heteroaromatic compounds aresubstituted benzothiazoles having the formula:

[0051] wherein X may be oxygen, NR⁷ or sulfur, wherein R⁷ is hydrogen oralkyl;

[0052] R¹ is missing or is a lower alkyl group;

[0053] R² is hydrogen, amino, optionally substituted lower alkyl, loweralkoxy, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted aralkyl, optionally substituted heteroaralkyl,optionally substituted lower alkylthio, optionally substituted arylthio,optionally substituted heteroarylthio,2-amino-α-(methoxyimino)-4-thiazolethiolacetate, guanidino, carboxy,alkylcarboxy, optionally substituted ureido, acetoacetamido,p-toluensulfonamidyl, or lower alkanoyloxy; and

[0054] R₃-R₆ independently is hydrogen, amino, halo, nitro, cyano,isocyano, hydroxy, sulfonyl, carboxy, optionally substituted alkyl,alkoxy, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted aralkyl, optionally substituted heteroaralkyl,optionally substituted lower alkylthio, optionally substituted arylthio,optionally substituted heteroarylthio, guanidino, optionally substitutedureido, or lower alkanoyloxy. Preferred R₃-R₆ groups include those whichimpart water solubility to the compounds. Also preferred are compoundswherein R¹ is missing.

[0055] Examples of optionally substituted heteroaromatic compounds thatmay be used in the practice of the invention include primuline,thioflavin S, 2-(4-aminophenyl)-6-methyl-7-sulfobenzothiazole, DirectYellow 8, Direct Yellow 9, Direct Yellow 27,S-2-benxothiazolyl-2-amino-α-(methoxyimino)-4-thiazolethiolacetate,3-(2-benzothiazolyl)-1-propanesulfonic acid (and its salts),3-(2-benzothiazolyl)-7-(diethylamino)coumarin,3-(2-benzimidazolyl)-7-(diethyl-amino)coumarin,3-(2-benzothiazolyl)-7-octadecyloxycourmarin,7-octadecyloxy-3-[3-(3-sulfopropyl)-2-benzothiazolylio]coumarin,3-(2-benzothiazolyl)umbelliferone, (2-benzothiazolyl)guanidine,2,6-dimethoxy-4-(2-benzothiazolyl)phenol, 2-(2-benzothiazolyl)ethanol,2-(4-aminophenyl)-6-methylbenzothiazole, 2-benzothiazole sulfide,2-benzothiazolylsulfonic acid (and its salts),3-(2-benzothiazolylamino)-5-nitrophenol,3-(2-benzothiazolyl)proprionitrile,4-(6-methyl-2-benzothiazolyl)acetanilide,4-(2-benzothiazolylthio)butyric acid (and its salts),4-(6-methyl-2-benzothiazolyl)phenyl isocyanate, luciferin, ethyl(2-benzothiazolylthio)formate, N-(2-benzothiazolyl)acetoacetamide,N-(2-methyl-6-benzothiazolyl)acetamide,N-(3-(2-benzothiazolyl)-4-hydroxy-phenyl)-p-toluenesulfonamide, andN-(6-nitro-2-benzothiazolyl)acetamide, many of which are available fromAldrich, Milwaukee, Wis.

[0056] Where the optionally substituted heteroaromatic compound issubstituted by a carboxy or sulfonyl group, the group may be in the formof a salt, e.g. the sodium, potassium or ammonium salt. In the matrix,the cation of the salt may be replaced with a positively charge sidechain from the protein. In addition, amino groups on aprotein/polypeptide may form coordination complexes with metals such assilver. However, since these amino groups also react with anionicmolecules added to the matrix, the ability of proteins/polypeptides tocoordinate to silver ions will be drastically altered. However, if theheteroaromatic compound contains an additional group capable of bindingsilver, the chelating property of the complex will be retained. Theformation of salt complexes between positive protein side chains ofproteins/peptides and the negative sulfonate substituted sensitizersleads to sensitivity advantages over other silver staining methods,particularly for low molecular weight proteins.

[0057] Many of the polyaminobenzothiazole sulfonic acid derivatives aredark colored materials. These materials are not suitable for silverstaining because of the residual dark color left in the matrix givingthe appearance of a heightened background. Thus, in a preferredembodiment, the optionally substituted heteroaromatic compound is notblack or very dark in color when in solution. Primuline and thioflavin Sare typically brown colored solids. However in concentrated solutions,they are orange and, when diluted, are a yellow color.

[0058] Examples of contrast enhancing agents include sodium sulfide,thiourea, dithiothreitol, potassium tetrathionate, sodium dithionite,and the sodium or potassium salt of thiosulfate. Sodium thiosulfate isknown to act as a contrast enhancing agent. (Wood, H. W., J. Phot. Sci.2: 154 (1954)). The silver deposited in the biopolymer within a matrixis more easily reduced in the presence of sulfur containing compounds.It is believed that silver sulfide acts as a catalyst for the reductionof silver ions. In a preferred embodiment, the contrast enhancing agentis present at a concentration of about 0.05% to 0.25%.

[0059] Examples of buffers that may be used in the practice of theinvention include known biological buffers with pKa's from about 5-10.See the Sigma Life Sciences Catalog, 2000-2001, pp. 962-965. Preferredbuffers are aqueous morpholinoethanesulfonic acid,morpholinopropanesulfonic acid (MOPS),4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and thelike.

[0060] A preferred sensitizing reagent comprises about 0.1 Mmorpholinoethanesulfonic acid (MES), about 5 mM sodium thiosulfate, andabout 0.025% primuline. The sensitizing reagent may further compriseabout 30% ethanol in water. Another preferred sensitizing reagentcomprises about 0.1 M MES, about 5 mM sodium thiosulfate pentahydrate,about 0.025 g/L thioflavin S, and about 1% dimethylformamide (DMF). Inorder to prepare this reagent, thioflavin S is dissolved in DMF and aninsoluble impurity is removed. This solution is then added to a solutionof MES and thiosulfate, titrated to pH 6.1. Although a saltprecipitates, it does not affect the performance of the reagent.Alternatively, thioflavin S, MES and thiosulfate may be dissolved inwater to give a solution without any DMF. This reagent is not preferredas it does not work as well when the reaction is heated with microwaveenergy. Putting DMF back into the solution restores the performance, butbrings back the precipitate.

[0061] Next, the matrix is contacted with a stainer solution comprisingone or more photosensitive or reduceable metal salts, optionally withactuating irradiation, to stain the biopolymer or form a stain image.Preferably, the reduceable metal salt has a reduction potential of about0.5 to about 1.9 volts. Examples of such reduceable metal salts includesilver, gold, platinum and/or iridium salts. Silver salts are preferredbecause they are less costly. Silver nitrate is a most preferredreduceable metal salt. Alternatively, a silver ammonia complex, e.g.prepared with silver nitrate and ammonium hydroxide, may be employed.The concentration of the salt in water is inversely (but notproportionally) dependent on the thickness of the matrix. Thus, anultrathin matrix of 0.001 mm or less requires higher concentrations ofthe reduceable metal salt(s), while thicker matrixes require lowerconcentrations. See U.S. Pat. No. 4,405,720. A typical concentration is0.1-0.2% silver nitrate in distilled water. The matrix is incubated fromabout 10-60 minutes with about 15 minutes being a preferred time. Whenmicrowave heating is employed, the incubation time may be reduced tofive minutes including the time of irradiation.

[0062] The use of light irradiation can be used when the matrixthickness is greater than about 1 mm or when staining certain organicpolymers such as DNA. The irradiation may be in any conventional mannerand must be sufficient to effect the photochemical reaction. Lightradiation from actinic through infrared and/or heat may be employed.When used, the irradiation is preferably during the first minutes oftreatment with the reduceable salt(s), to produce maximum sensitivity.Typically, a bright uniform light source such as a 160 watt fluorescentgrid lamp which emits light equivalent to a 1,500 watt tungsten sourcemay be used. One of ordinary skill in the art can select an optimumlight source and time of irradiation for a given matrix,protein/polypeptide and nucleic acid molecule with no more than routineexperimentation.

[0063] Next the matrix is quickly rinsed with water to remove the freereduceable metal salt(s). Since silver binding is reversible the time ofrinsing should be kept to about 1 minute or less.

[0064] Next, the stain image is developed. This may be accomplished bycontacting the matrix with a developer solution comprising one or morereducing agents. Such reducing agents include any that are used inphotography including metallic compounds of iron, tungsten, vanadium andmolybdenum, and organic compounds including hydroquinone, pyrogallol,p-phenylenediamine, paraformaldehyde and formaldehyde. Organic reducingreagents are preferred. When formaldehyde or paraformaldehyde is thereducing agent, it is preferred to further add an alkalinizing agentsuch as sodium carbonate or sodium metaborate to the solution. Since thelatent image development may continue until the matrix is contacted withthe reducing agent(s), one may immerse the matrix immediately into thereducing agent as soon as it is removed from the reduceable metal saltcontaining solution.

[0065] In a preferred embodiment, the matrix is immersed in a basicbuffer solution whose pH is between 11 and 12 and which containsformaldehyde. Preferred buffers include sodium and potassium carbonate.The immersion may be repeated as necessary or desired. Optionally, thematrix may be subjected to gentle agitation. A most preferred developersolution consists of 2.5% potassium carbonate and 0.35% formaldehyde(37% by weight) in distilled water. The development time is from about3-30 minutes depending upon the thickness of the gel, the extent ofsample loading and background staining intensity. One of ordinary skillin the art can determine the optimum development time with no more thanroutine experimentation.

[0066] Finally, the reaction in the matrix is stopped with a stoppersolution. This may be accomplished by thorough washing with water and/orby lowering the pH of the developer to between 7-10 and complexation ofthe unreduced silver with a chelating agent. For convenience a stoppersolution comprising a buffer or an acid and chelating agent may be addeddirectly to the developer solution. Examples of acids that may be usedinclude aqueous solutions of acetic, citric and hydrochloric acid.Examples of chelating agents include ethylenediamine tetraacetic acid. Apreferred chelation agent that also functions as a buffer isethylenediamine tetraacetic acid and its salts. A preferred stoppersolution is 0.5 M ethylenediamine tetraacetic acid trisodium salt, pH 8.Typically the matrix is submerged in the stopper solution for tenminutes, optionally with gentle agitation, and then washed withdistilled water. One of ordinary skill in the art can determine optimumtime and conditions for stopping development of the latent image with nomore than routine experimentation.

[0067] Once the stain image has been developed, it can be detected byany conventional means for detection including visual inspection,scanning, e.g. with a conventional flatbed scanner, or with an imagingcamera. See, for example, U.S. Pat. No. 4,703,016.

[0068] The invention also relates to a method for identifying a proteinor peptide in a matrix, comprising:

[0069] (a) contacting the matrix with a sensitizing reagent comprisingone or more optionally substituted heteroaromatic compounds;

[0070] (b) contacting the matrix with one or more reduceable metal saltsto stain said protein or peptide;

[0071] (c) detecting the stained protein or peptide and thereby theprotein or peptide;

[0072] (d) carrying out a cleavage reaction on the protein or peptide togive fragments; and

[0073] (e) carrying out a mass spectrometric analysis on said fragmentsthereby identifying the protein or peptide.

[0074] In carrying out this method, the protein or peptide may be firstfixed to the matrix as described herein. In a preferred embodiment, theprotein or peptide is fixed to the matrix by contacting the matrix witha fixing reagent consisting of ethanol, acetic acid and water.

[0075] Before the stain protein or peptide is detected, it may betreated with a developing solution as described herein which comprisesone or more reducing agents. The stain image may then be detectedaccording to any of the methods described herein.

[0076] In order to carry out a cleavage reaction on the protein orpeptide, the stain protein or peptide may be excised from the matrix andthe protein or peptide isolated from the matrix. For example, theprotein or peptide may be excised from the matrix with a scalpel andplaced into a container. In a preferred embodiment, an aqueous mixtureof potassium ferricyanide and sodium thiosulfate is added to thecontainer to reoxidize substantially all of the reduced silver metalgrains to silver ions and complex them to thiosulfate. The protein orpolypeptide may be extracted with water from one up to six or moretimes. The extracts may then be treated with a cleavage reagentcomprising a protease such as trypsin, and the fragments subjected tomass spectrometric analysis to determine identity. See Shevenko, A. etal., Anal. Chem. 68:850-858 (1996); Helmannn, V. et al., Anal. Biochem.224:451-455 (1995); Coligan, J. E. et al., in Current Protocols inProtein Science, V. B. Chanda (ed.) (1998); Patterson, S. D. andAebersold, R., Electrophoresis 16:1791-1814 (1995); and Bergman et al.,Electrophoresis 21:679-686 (2000). As discussed above, this method is animprovement over prior methods using glutaraldehyde as no cross linkingof the proteins and polypeptides occur. In addition, the sensitizingreagent provides for the sensitive detection of the proteins andpolypeptides without the need for glutaraldehyde.

[0077] The present invention is a great advance in the art in that a newclass of sensitizing agents has been discovered. These new agents bindto the biopolymers that have been fixed within a matrix and increase thelocalized binding of the reduceable metal salt to the biopolymer zone.These sensitizing agents only bind the reduceable metal salt ion weaklyso that reduction of the ions may occur when the matrix is subjected toreducing conditions as described herein.

[0078] The increase in sensitivity for biopolymers achieved according tothe present invention is believed to result from several factorsdiscussed below. First, while not wishing to be bound by any particulartheory, it is believed that the charge—charge attraction of the sulfonicacid groups of the thioflavin S with positively charged side chains andpossibly interactions of the aromatic benzothiazole ring withhydrophobic regions of the protein structure allow binding of thesensitizer to the proteins within the matrix. In addition, it isbelieved that the sulfur and nitrogen atoms of the benzothiazole ringstructure are capable of forming weak coordination complexes withsilver(+1) ions which can then be reduced efficiently to silver metalwith a dilute formaldehyde solution at a pH>10. For nucleic acids, it isbelieved that the mechanism is more likely an intercalation effect inwhich the planar aromatic nucleus of the benzothiazole ring structurealigns between two adjacent pyrimidine or purine bases to form a complexwhich is stabilized by pi stacking interactions. Again the silverbinding occurs through the sulfur and/or nitrogen of the benzothiazolering system and is bound weakly enough to be reduced by formaldehyde inalkali medium above pH 10.

[0079] The invention also relates to a kit for the detection ofbiopolymers, comprising one or more components selected from the groupconsisting of

[0080] (a) a sensitizing reagent comprising one or more optionallysubstituted heteroaromatic compounds;

[0081] (b) one or more reduceable metal salts;

[0082] (c) one or more developer solutions comprising a reducing agent;

[0083] (d) one or more stopper solutions which prevent further reductionof the reduceable metal salts;

[0084] (e) one or more buffers;

[0085] (f) one or more fixing reagents;

[0086] (g) one or more cleaving reagents;

[0087] (h) one or more biopolymers;

[0088] (i) one or more matrixes; and

[0089] (j) one or more indicators which are sensitive to pH changes.

[0090] The kit in general is a carton, box, tube or the like andcontains one or more containers, each of which containing one or more ofthe components listed above. Such containers include boxes, bottles,jars, tubes, ampoules and the like.

[0091] Biopolymers may be included in the kit for use, for example, asstandards or controls.

[0092] Examples of matrixes that may be included in the kit includepolyacrylamide gel, agarose, paper, cellulose acetate, ornitrocellulose.

[0093] Examples of indicators that may be included in the kit includephenolphthalein and thymolphthalein.

[0094] The invention also relates to compositions for carrying out themethods of the present invention and to compositions made when carryingout the invention. Such compositions may comprise one or more componentsselected from the group consisting of

[0095] (a) a sensitizing reagent comprising one or more optionallysubstituted heteroaromatic compounds;

[0096] (b) one or more reduceable metal salts;

[0097] (c) one or more developer solutions comprising a reducing agent;

[0098] (d) one or more stopper solutions which prevent further reductionof the reduceable metal salts;

[0099] (e) one or more buffers;

[0100] (f) one or more fixing reagents;

[0101] (g) one or more cleaving reagents;

[0102] (h) one or more biopolymers;

[0103] (i) one or more matrixes; and

[0104] (j) one or more indicators which are sensitive to pH changes.

[0105] The invention also relates to the compositions obtained by thepractice of the invention. Such compositions include, withoutlimitation, the various reaction mixtures that are obtained as well asthe cleaved proteins and peptides obtained with a cleaving reagent.

[0106] The following examples are illustrative, but not limiting, of themethod and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in the art and which are obvious tothose skilled in the art are within the spirit and scope of theinvention.

EXAMPLES Example 1

[0107] Basic Staining Procedure

[0108] In the following example, the formulations were made up asfollows. All solutions were made w/ultrapure water. Sensitizer: 1 MMorpholinoethane sulfonic acid 50 mM Sodium thiosulfate pentahydrate0.25 g/L Thioflavin S 10% Dimethylformamide Stainer: 20% Silver nitrateDeveloper: 15% Potassium carbonate 0.075% Sodium thiosulfatepentahydrate 0.002% Phenolphthalein Developer Enhancer: 37% FormaldehydeStopper Solution: 0.5 M Ethylenediamine tetraacetic acid 1.5 MTris(hydroxymethyl)aminomethane Destainer A: 30 mM PotassiumFerricyanide Destainer B: 100 mM Sodium thiosulfate pentahydrate

[0109] Basic Staining Protocol

[0110] Before starting, prepare the following solutions for staining:*Sensitizing solution Ethanol 30 ml Sensitizer 10 ml Ultra pure water to100 ml *Staining solution Stainer 1 ml Ultra pure water to 100 ml*Developing solution Developer 10 ml Developer enhancer 1 drop Ultrapure water to 100 ml

[0111] The solutions may be prepared immediately before starting thestaining protocol or as one proceeds to the next step.

[0112] Procedure

[0113] This procedure is for use with an 8×8 cm NuPAGE® Bis-Tris,Tris-Acetate, TBE or Tris-Glycine mini-gel, 1.0 mm thick. If one stainstwo mini-gels, 1.0 mm thick or one large (18×18 cm) gel, all solutionvolumes should be doubled while maintaining the same incubation time.One may have to optimize the staining protocol, if the dimensions of thegel are not the same as mentioned above.

[0114] All incubations should be performed on a rotary shaker rotatingat a speed of approximately 1 revolution/sec at room temperature. Thereshould be 100 ml of solution per gel.

[0115] 1. After electrophoresis, remove the gel from the cassette andplace it in a clean staining tray of the appropriate size. Optionally,the gel may be rinsed twice with ultra pure water.

[0116] 2. Fix the gel in 100 ml of fixative for 60 minutes with gentlerotation.

[0117] (Note: The gel can be stored in the fixative overnight if thereis not enough time to complete the staining protocol.)

[0118] 3. Decant the fixative solution and wash the gel in 100 ml of 30%ethanol for 10 minutes.

[0119] 4. Add 100 ml of Sensitizing solution to the washed gel in thestaining container. Incubate the gel in the Sensitizing solution for 10minutes.

[0120] 5. Wash the gel in 100 ml of 30% ethanol for 10 minutes.

[0121] 6. Decant the Sensitizing solution and wash the gel in 100 ml ofultra pure water for 10 minutes.

[0122] 7. Place the gel in 100 ml of Staining solution for 15 minutes.

[0123] 8. After staining is complete, decant the Staining solution andwash the gel with 100 ml of ultra pure water for 1 minute.

[0124] Note: Washing the gel for more than a minute will remove silverions from the gel and result in decreased sensitivity.

[0125] 9. Incubate the gel in 100 ml of Developing solution for 5-8minutes until bands start to appear.

[0126] 10. Once the desired band intensity is achieved, immediately add10 ml of Stopper directly to the gel still immersed in Developingsolution. Gently agitate the gel for 10 minutes. The color changes frompink to colorless indicating that the development has stopped.

[0127] 11. Decant the Stopper solution and wash the gel with 100 ml ofultra pure water for 10 minutes.

[0128] If one is to destain the gel for mass spectrometry analysis, seeExample3.

EXAMPLE 2 Fast Staining Procedure

[0129] Introduction

[0130] The fast staining protocol is a modification of the basicstaining protocol. This method uses a microwave oven to rapidly silverstain protein gels. This staining protocol can be completed in less thanan hour.

[0131] Materials Needed

[0132] Ultra pure water

[0133] Microwaveable staining tray

[0134] Microwave

[0135] Rotary shaker

[0136] Teflon coated stir bars

[0137] Disposable 10 ml pipettes

[0138] Clean glass bottles for reagent preparation

[0139] Graduated Glass Cylinders

[0140] 30% ethanol (made with ultra pure water)

[0141] 100% ethanol

[0142] Fixative (40% ethanol, 10% acetic acid, made with ultra purewater)

[0143] Before Starting

[0144] Prepare the following solutions for staining: *Sensitizingsolution Ethanol 30 ml Sensitizer 10 ml Ultra pure water to 100 ml*Staining solution Stainer 1 ml Ultra pure water to 100 ml *Developingsolution Developer 10 ml Developer enhancer 1 drop Ultra pure water to100 ml

[0145] Procedure

[0146] For use with an 8×8 cm NuPAGE® gel, 1.0 mm thick. Be sure to have100 ml of solution per gel. (Note: One may have to optimize the stainingprotocol, if the dimensions of your the are not the same as mentionedabove.)

[0147] 1. After electrophoresis, remove the gel from the cassette andplace it in a clean microwaveable staining tray of appropriate size.Rinse the gel twice with ultra pure water.

[0148] 2. Place the gel in 100 ml of fixative and microwave at highpower for 30 seconds. Remove the gel from the microwave and gentlyagitate it for 5 minutes at room temperature. Decant the fixative.

[0149] 3. Wash the gel with 100 ml of 30% ethanol in a microwave at highpower for 30 seconds. Remove the gel from the microwave and gentlyagitate it for 5 minutes at room temperature on a rotary shaker.

[0150] 4. Add 100 ml of Sensitizing solution to the washed gel.Microwave the gel at high power for 30 seconds. Remove the gel from themicrowave and place it on a rotary shaker for 2 minutes at roomtemperature. Decant the sensitizing solution.

[0151] 5. Wash the gel twice in 100 ml ultra pure water. Microwave athigh power for 30 seconds. Remove the gel from the microwave and gentlyagitate it for 2 minutes at room temperature. Decant the water.

[0152] 6. Place the gel in 100 ml of Staining solution. Microwave athigh power for 30 seconds. Remove the gel from the microwave and gentlyagitate it for 5 minutes at room temperature.

[0153] 7. Decant the Staining solution and wash the gel with 100 ml ofultra pure water for 1 minute.

[0154] 8. Place the gel in 100 ml of Developing solution and incubatefor 5 minutes with gentle agitation on a rotary shaker. (Do notmicrowave).

[0155] 9. Once the desired band intensity is achieved, immediately add10 ml of Stopper directly to the gel still immersed in Developingsolution and gently agitate the gel for 10 minutes. The color changesfrom pink to clear indicating that the development has stopped.

[0156] 10. Wash the gel with 100 ml of ultra pure water for 10 minutes.

[0157] 11. If one must destain the gel for mass spectrometry analysis,see Example 3.

EXAMPLE 3 Destaining Procedure for Mass Spectrometric Analysis

[0158] When preparing samples for mass spectrometry analysis, it isimportant to remove silver ions from protein bands before performingin-gel trypsin digestion (Gharahdaghi et al., Electrophoresis 20:601-605(1999)). A destaining protocol to effectively remove silver ions fromthe gel is described below.

[0159] Materials Needed:

[0160] Clean scalpel

[0161] 1.5 ml sterile microcentrifuge tubes

[0162] Ultra pure water

[0163] Microcentrifuge

[0164] Procedure

[0165] For use with NuPAGE® Bis Tris, Tris-Glycine or Tris-Acetate 8×8mini-gels, 1.0 mm thick.

[0166] 1. After silver staining of the gel, wash the gel thoroughly withultra pure water.

[0167] 2. Carefully excise the band(s) of interest using a clean scalpeland place into 1.5 ml sterile microcentrifuge tube(s). Excise anotherpiece of gel of the same size from a blank region of the gel and placeinto another sterile microcentrifuge tube. This will be used later as acontrol for trypsin digestion.

[0168] 3. Add 50 μl of Destainer A and 50 μl of Destainer B to eachmicrocentrifuge tube.

[0169] Note: If destaining a large number of gel bands, then prepare therequired amount of the destaining solution by mixing Destainer A and B,and use immediately. Destainer solutions A and B cannot be stored forlong periods once they are mixed.

[0170] 1. Mix the contents of the tube thoroughly and incubate for 15minutes at room temperature.

[0171] 2. Carefully remove the supernatant using a clean pipette tip.

[0172] 3. Add 200 μl of ultra pure water to the tube and mix thoroughly.Incubate for 10 minutes at room temperature.

[0173] Repeat steps 5-6 at least two times. Proceed to trypsin digestionand analysis by mass spectrometry.

[0174] Having now fully described this invention, it will be understoodby those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

What is claimed is:
 1. A method for detecting a biopolymer in a matrix,comprising: (a) contacting the matrix with a sensitizing reagentcomprising one or more optionally substituted heteroaromatic compounds;(b) contacting the matrix with one or more reduceable metal salts tostain said biopolymer; and (c) detecting the stained biopolymer.
 2. Themethod of claim 1, wherein said matrix is a polyacrylamide gel, agarose,paper, cellulose acetate, or nitrocellulose.
 3. The method of claim 1,wherein said biopolymer is fixed to the matrix by treatment of thematrix with a fixing reagent comprising an aqueous solution of anorganic acid and a lower alcohol containing 1-4 carbon atoms.
 4. Themethod of claim 3, wherein said lower alcohol is methanol, ethanol,propanol, or isopropanol.
 5. The method of claim 3, wherein said organicacid is acetic acid, citric acid, sulfosalicylic acid or trichloroaceticacid.
 6. The method of claim 3, wherein said fixing reagent consists ofabout 40% ethanol, about 10% acetic acid and about 50% distilled waterby volume.
 7. The method of claim 1, wherein said substitutedheteroaromatic compound is substituted with a group which imparts watersolubility.
 8. The method of claim 1, wherein said optionallysubstituted heteroaromatic compound is primuline, thioflavin S or2-(4-aminophenyl)-6-methyl-7-sulfobenzothiazole.
 9. The method of claim1, further comprising contacting said matrix with said sensitizingreagent together with one or more contrast enhancing agents and one ormore buffers.
 10. The method of claim 9, wherein said contrast enhancingagents are selected from the group consisting of sodium sulfide,thiourea, dithiothreitol, potassium tetrathionate, sodium dithionite,and the sodium or potassium salt of thiosulfate.
 11. The method of claim9, wherein said one or more buffers has a pKa of 5-10.
 12. The method ofclaim 9, wherein said one or more buffers is aqueousmorpholinoethanesulfonic acid.
 13. The method of claim 1, wherein saidone or more reduceable metal salts is silver nitrate.
 14. The method ofclaim 1, wherein said stained biopolymer is developed prior to saiddetecting by contacting the matrix with one or more reducing agents. 15.The method of claim 14, wherein said one or more reducing agents isformaldehyde.
 16. The method of claim 1, wherein said stained biopolymeris detected visually.
 17. The method of claim 1, wherein said stainedbiopolymer is detected by scanning the image with an electronic scanner.18. The method of claim 1, wherein said stained biopolymer is detectedwith an imaging camera.
 19. The method of claim 1, wherein said matrixis heated when contacted with at least one of the sensitizing reagentand the one or more reduceable metal salts.
 20. The method of claim 19,wherein said heating is carried out with microwave energy.
 21. Themethod of claim 1, wherein said biopolymer is a protein.
 22. The methodof claim 1, wherein said biopolymer is a peptide.
 23. The method ofclaim 1, wherein said biopolymer is a nucleic acid molecule.
 24. Amethod for detecting a protein/peptide or nucleic acid molecule in apolyacrylamide gel, comprising: (a) fixing the protein/peptide ornucleic acid molecule to the polyacrylamide gel; (b) contacting thepolyacrylamide gel with a sensitizing reagent comprising primuline,thioflavin S or 2-(4-aminophenyl)-6-methyl-7-sulfobenzothiazole; (c)contacting the polyacrylamide gel with an aqueous solution of a silversalt to stain the protein/peptide or nucleic acid molecule, (d)developing the stained protein/peptide or nucleic acid molecule, and (e)detecting the stained protein/peptide or nucleic acid molecule.
 25. Themethod of claim 24, wherein said protein/peptide or nucleic acidmolecule is fixed to the polyacrylamide gel by contacting thepolyacrylamide gel with a fixing reagent consisting of about 40%ethanol, about 10% acetic acid and about 50% distilled water by volume.26. The method of claim 24, wherein said sensitizing agent furthercomprises aqueous morpholinoethanesulfonic acid and one or more contrastenhancing agents selected from the group consisting of sodium sulfide,thiourea, dithiothreitol, potassium tetrathionate, sodium dithionite,and the sodium or potassium salt of thiosulfate.
 27. The method of claim24, wherein said stained protein/peptide or nucleic acid molecule isdeveloped by contacting the polyacrylamide gel with aqueousformaldehyde.
 28. A method for identifying a protein or peptide in amatrix, comprising: (a) contacting the matrix with a sensitizing reagentcomprising one or more optionally substituted heteroaromatic compounds;(b) contacting the matrix with one or more reduceable metal salts tostain said protein or peptide; (c) detecting the stained protein orpeptide; (d) carrying out a cleavage reaction on the protein or peptideto give fragments; and (e) carrying out a mass spectrometric analysis onsaid fragments thereby identifying the protein or peptide.
 29. A kit forthe detection of biopolymers, comprising one or more components selectedfrom the group consisting of: (a) a sensitizing reagent comprising oneor more optionally substituted heteroaromatic compounds; (b) one or morereduceable metal salts; (c) one or more developer solutions comprising areducing agent; (d) one or more stopper solutions which prevent furtherreduction of the reduceable metal salts; (e) one or more contrastenhancing agents; (f) one or more buffers; (g) one or more fixingreagents; (h) one or more cleaving reagents; (i) one or more biopolymers(j) one or more matrixes; and (k) one or more indicators which aresensitive to pH changes.
 30. A composition, comprising one or morecomponents selected from the group consisting of: (a) a sensitizingreagent comprising one or more optionally substituted heteroaromaticcompounds; (b) one or more reduceable metal salts; (c) one or moredeveloper solutions comprising a reducing agent; (d) one or more stoppersolutions which prevent further reduction of the reduceable metal salts;(e) one or more contrast enhancing agents; (f) one or more buffers; (g)one or more fixing reagents; (h) one or more cleaving reagents; (i) oneor more biopolymers; (j) one or more matrixes; and (k) one or moreindicators which are sensitive to pH changes.
 31. The composition ofclaim 30, wherein said one or more indicators is phenolphthalein orthymolphthalein.
 32. The composition of claim 30, wherein said one ormore fixing reagents comprises an aqueous solution of an organic acidand a lower alcohol containing 1-4 carbon atoms.
 33. The composition ofclaim 32, wherein said lower alcohol is methanol, ethanol, propanol, orisopropanol.
 34. The composition of claim 32, wherein said organic acidis acetic acid, citric acid, sulfosalicylic acid or trichloroaceticacid.
 35. The composition of claim 30, wherein said one or more fixingreagents consists of about 40% ethanol, about 10% acetic acid and about50% distilled water by volume.
 36. The composition of claim 30, whereinsaid optionally substituted heteroaromatic compound is primuline,thioflavin S or 2-(4-aminophenyl)-6-methyl-7-sulfobenzothiazole.
 37. Thecomposition of claim 30, wherein said one or more contrast enhancingagents are selected from the group consisting of sodium sulfide,thiourea, dithiothreitol, potassium tetrathionate, sodium dithionite,and the sodium or potassium salt of thiosulfate.
 38. The composition ofclaim 30, wherein said one or more buffers has a pKa of 5-10.
 39. Thecomposition of claim 30, wherein said one or more buffers ismorpholinoethanesulfonic acid,4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), or4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (HEPES).
 40. Thecomposition of claim 30, wherein said one or more reducible metal saltsis silver nitrate.